Solvent extracted raw karanj cake (rSKC), concentrate mixture and substrate for in vitro study
Solvent extracted raw karanj cake (rSKC) was supplied by Ayurvet Pvt. Ltd., New Delhi, India. Different levels of rSKC was incorporated replacing 0 (rSKC-0, control), 25.0 (rSKC-25), 50.0 (rSKC-50), 75.0 (rSKC-75) and 100 (rSKC-100) percent CP moiety of soybean meal in the concentrate mixture (22% CP) as protein source. The ingredient composition of the concentrate mixtures containing different levels of rSKC is presented in Table 1. The concentrate mixture and finely grounded wheat straw (1: 1 on weight basis) were used as substrate for evaluation by
in vitro gas production technique
(Menke et al., 1979; Menke and Steingass 1988).
Chemical analysis of solvent extracted raw karanj cake (rSKC)
Solvent extracted raw karanj cake (rSKC) was analyzed for proximate principles
AOAC (2005) to determine DM by the oven drying method, organic matter (OM) and total ash by muffle furnace incineration, crude protein (CP) by Kjeldahl method and ether extract (EE) by Soxhlet apparatus. Crude fibre (CF) was analysed by refluxing the feed sample with weak acid and alkali. NFE was obtained by calculation and added with CF to arrive at total carbohydrates. Neutral detergent fibre (NDF) and acid detergent fibre (ADF) were determined by the methods of Van
Soest et al., (1991).
Toxic principles of solvent extracted raw karanj cake (rSKC)
For karanjin and pongamol estimation, methanol extracts of rSKC was prepared and concentrated under vacuum using rota-vac (Heidolph Instruments, Schwalbach, Germany). Estimation of karanjin from rSKC was carried out by the method described by
Ravikanth et al., (2009) using High Performance Thin Layer Chromatography (HP-TLC; CAMAG, Switzerland). Similarly, pongamol was also estimated using HP-TLC (CAMAG, Switzerland) with hexane: ethyl acetate (85:15) solvent system using D
2 lamp scannerand detector at 320 nm absorbance
(Kumar et al., 2011). Trypsin inhibitor activity in KC was estimated spectro photometrically as per
Roy and Rao (1971) using casein as a substrate for assaying the activity of the trypsin enzyme.
Donor animals for rumen liquor
Two fistulated adult bulls (B.Wt 400 kg approx), aged about 3 years were used for collection of rumen liquor for
in vitro studies. The animals were fed wheat straw
ad libitum and concentrate mixture (Maize, 25; Soybean meal, 35; Wheat bran, 38; Mineral mixture, 1.0 and Salt 1.0 percent) in the morning (09.30 h) to meet their nutrient requirement for maintenance (
Kearl, 1982). Clean drinking water was provided free choice to all the animals. The animals were housed in well-ventilated shed with provision of individual feeding.
Weighing of substrates and greasing of syringes
The finely ground feed samples (1.0 mm screen sieve) were weighed on a weighing boat with removable stem without leaving sample sticking on its side. The samples in the required proportion (Conc. mixture and wheat straw, 50:50) as per treatment were introduced at the bottom of syringes. After weighing, the piston was greased with paraffin soft white LR (S.D. Fine-Chem. Ltd.; M.P. 39-56°C) and pushed into the barrel of the syringe.
Preparation of buffer media
All the solutions were prepared as per
Menke and Steingass (1988). The solutions were poured into a Woulff flask, mixed with a magnetic stirrer and warmed to 39°C in a PVC water bath with digital thermostat (Julabo Labortechnik GmBH Seelbach, Germany) in the specified order. Carbon dioxide gas was passed through the submerged tube in the Woulff flask continuously during buffer media preparation. On the day of incubation, the mixture of rumen liquor and particulate matter (approximately 60:40) was collected from the fistulated donor animal (before feeding) into pre-warmed CO
2 filled thermos flask and carried to the laboratory. The rumen fluid was bubbled with CO
2 gas for few minutes and then mixed in a laboratory blender at medium speed to remove microbes attached to particulate matter. Rumen liquor was then strained through a double layer of muslin cloth. Strained liquor was added to the buffer media when the media became colorless. All handling of rumen liquor was done under continuous flushing with CO
2.
Estimation of total gas production
The buffered rumen fluid (30ml) was dispensed to each syringe by an automatic dispenser (OPTIFIX, Walter Graf and Co., Wertheim). After recording initial volume (± 0.5 ml), the syringes were placed in the incubator maintained at 39°C. The syringes were shaken by hand intermittently. If the gas volume exceeded 80 ml mark, gas was released and the incubation was continued. All incubations were run in triplicate and four syringes with buffered rumen fluid were incubated as blanks. At the end of incubation (24 h) the amount of gas produced was recorded by noting displacement of the piston of syringe and the contents of the syringes were analyzed further.
Determination of substrate degradation and microbial bio-mass production
The contents of the syringes were transferred to 500 ml spout-less beakers by repeated washings with neutral detergent solution without sodium sulphate (
Van Soest and Robertson, 1985). The contents were then refluxed for 1 h and the residue was recovered in pre-weighed filter crucibles. After drying the crucibles (with residue) to constant weight, ashing was done at 450°C for 30 minutes. Truly degradable organic matter in rumen (TDOMR) and microbial biomass production (MBP) were calculated as follows:
TDOMR = Feed (OM) incubated - residue (OM)
MBP = TDOMR - (2.2 x net gas volume)
EMP = {TDOMR - (2.2 x net gas volume)} x 100/TDOMR
PF = TDOMR/net gas volume
Statistical analysis
The experimental data generated were analyzed by using one way ANOVA (
Snedecor and Cochran, 1989) and
post hoc tests through statistical package SPSS 19.0. Treatment means were ranked using Duncan’s multiple range test and expressed with standard error of mean (SEM). Significance of treatments with respect to different characters was declared at P≤0.05 unless otherwise stated.
